Dissecting the role of matrix metalloproteinases (MMP) and integrin αvβ3 in angiogenesis in vitro: Absence of hemopexin C domain bioactivity, but membrane-type 1-MMP and α vβ3 are critical

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin αvβ 3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic Inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but mot TIMP-1. TMs confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin αvβ3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin αvβ3. Moreover, no specific binding of pro-MMP-2 to integrin αvβ3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin αvβ3, and this in a PEX-independent manner. Likewise, integrin αvβ 3-expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and αvβ3-dependent but MMP-2 independent and does not support a role for PEX in αvβ3 integrin binding or in modulating angiogenesis in this system. ©2005 American Association for Cancer Research.